Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

• Published in: The journal of physical chemistry. B Vol. 119; no. 8; pp. 3356 - 3365
• Main Authors: Han, Yang, Hsu, Julie, Ge, Nien-Hui, Potma, Eric O
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.02.2015
• Subjects: Animals; Anisotropy; Avian Proteins - chemistry; Collagen - chemistry; Cornea - chemistry; Hawks; Microscopy - methods; Rats; Spectrum Analysis; Tail - chemistry; Tendons - chemistry; Vibration
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp511058b

Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm–1 stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging. Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagen’s second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ρ of the signal for SFG (ρ ≈ 15) compared to SHG (ρ ≈ 3). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.